微量小鼠小脑粒细胞cDNA文库构建和快速鉴定

cDNA LIBRARY CONSTRUCTION AND QUICK ASSAY FROM MOUSE GRANULE CELLS

应用一步RNA提取法,改进经典cDNA文库构建程序,并用PCR技术鉴定cDNA文库的有效容量和插入cDNA的大小。从200mg小鼠小脑粒细胞中建立起容量为7×10~6个重组子的cDNA文库,为进一步研究小鼠缺陷株WEVER型的粒细胞丢失原因和基因表达缺陷提供正常小鼠粒细胞的cDNA文库。

It was believed that there were some gene-expressing deficiencies in WEVER mouse granule cells.We constructed the cDNA library from the normal mouse granule cells,so as to supply the candidate genes for further screening and isolating the target genes related to WEVER mouse. With only 200 mg granule cells,one step RNA extraction was used to yield high quantity and quality RNA in stead of CsCl ultrocentrifuge method.The modified library procedures reduced the cDNA lost to a minimum.7×10~6 recombinants were packaged in lamda gtll phage,which contained 200bp to 7.2kb insert cDNA tested by PCR technique.Dot blot presented a high homology between PCR cDNA and granule cDNA probes.